The Structure of Monosodium Phosphoenolpyruvate

Transcription

1 834 STRUCTURE OF DIAQUA(N-SALICYLIDENE-L-THREONINATO)COPPER(II) KORHONEN, K. & H.AM.AL.~.INEN, R. (1979). Acta Chem. Scand. Ser. A, 33, MAIN, P., WOOLFSON, M. M., LESSINGER, L., GERMAIN, G. & DECLERCQ, J. P. (1974). MULTAN 74. A System of Computer Programs for the A utomatic Solution of Crystal Structures from X-ray Diffraction Data. Univs. of York, England, and Louvain, Belgium. STEWART, J. M., MACHIN, P. A., DICKINSON, C., AMMON, H. L., HECK, H. & FLACK, H. (1976). The XRAY system - version of Tech. Rep. TR-446. Computer Science Center, Univ. of Maryland, College Park, Maryland. UEKI, T., ASHIDA, T., SASADA, Y. & KAKUDO, i. (1967). Acta Cryst. 22, UEKI, T., ASHIDA, T., SAS~DA, Y. & KAKUDO, M. (1969). Acta Cryst. B25, Acta Cryst. (1981). B37, The Structure of Monosodium Phosphoenolpyruvate BY S. K. KATrI, M. V. HOSUR AND M. A. VISWAMITRA Department of Physics and ICMR Centre on Genetics and Cell Biology, Indian Institute of Science, Bangalore , India (Received 25 September 1980; accepted 3 November 1980) Abstraet CDH406P-.Na +.H20, M r = 208.0, is monoclinic, Cc, a = (2), b = (5), c (1) A, fl = (1), U = A 3, D x = Mg m -a, Z = 8, 2(Mo Ka) = A, p(mo Ka) = 0.44 mm -~, F(000) = 840. Final R = for 1697 reflections. The two crystallographically independent molecules of phosphoenolpyruvate (PEP) (A and B) are almost mirror images of each other, the mirror being the planar enolpyruvate group. The torsion angle C(3)-C(2)- O(1)-P(1) is in A and in B, in contrast to in PEP.K. The enolic C(2)-O(1) has a partial double-bond character [1.401 (A), 1.386A (B)]. The high-energy P~O bond (1.595 and 1.610A) is comparable to that in PEP.K (1.612 A). Na(1) has six nearest neighbours while Na(2) has only five. The Na + ions are involved in binding only the phosphates of different molecules, in contrast to the K ion in PEP. K, which binds to both the phosphate and carboxyl ends of the same molecule. The planar carboxyl groups stack on each other at an average distance of 3.2 A instead of forming hydrogen-bonded dimers usually found in carboxylate structures. Introduction Phosphoenolpyruvate (PEP) is known to play an important role in glycolysis as a substrate in the pyruvate kinase catalysed reaction, where adenosine 5'-triphosphate is synthesized by a phosphate-group transfer from PEP to adenosine 5'-diphosphate. For this phosphorylation reaction to proceed, the presence of divalent Mg 2+ and monovalent K + cations is /81/ obligatory (Mahler & Cordes, 1969). Replacement of K + by Na + is known to inhibit the reaction. Hence it would be of interest to study the structure of PEP when bound to Na and compare it with that of its K + complex. We report here the structure of monosodium phosphoenolpyruvate (PEP.Na) monohydrate as obtained from single-crystal X-ray analysis. The conformation of PEP in the present structure is found to be significantly different from that in its K salt (Hosur & Viswamitra, 1981). Experimental Crystals of the monosodium salt of PEP (original sample from Boehringer Mannheim GmbH) were grown by slow diffusion of acetone into an aqueous solution of the compound. A single crystal 0.15 x 0.17 x 0.22 mm, with well developed prismatic faces, was used to determine the crystal data by rotation and Weissenberg photographs. The cell parameters were later refined on a CAD-4 diffractometer by least squares from 25 high-angle reflections. The density, measured by flotation in bromoform and acetone mixtures, indicated eight (PEP.Na.H20) units in the cell. Systematic absences hkl: h + k -- 2n + 1 and hol: l -- 2n + 1 indicated space groups Cc or C2/c. Mo Ka intensities to a 20-limit of 56 (resolution = 0.76 A) were collected on the diffractometer in the 09/20 scan mode. Background counts were measured at the two edges of the Bragg peak for ~ of the total scan angle. There was no significant change in the intensities of the monitor reflections checked at regular intervals. The data consisted of 1697 unique reflections in the hkl 1981 International Union of Crystallography

2 S. K. KATTI, M. V. HOSUR AND M. A. VISWAMITRA 835 and hki octants. These were corrected for Lorentz and polarization effects but not for absorption (,ur ~ 0.08). Structure solution and refinement Initial attempts to solve the structure in C2/c were unsuccessful. The structure was solved in Cc by the application of direct methods and difference syntheses with SHELX 76 (Sheldrick, 1976). The phosphates of the two crystallographically independent molecules were identified in the E map only after renormalizing the E values for l = 2n + 1 reflections. A difference synthesis based on these two phosphates revealed the positions of enolpyruvate atoms and the Na + ions. Block-diagonal least-squares refinement with individual isotropic temperature factors reduced R to and later to with the introduction of anisotropy for the thermal parameters. The two water molecules in the asymmetric unit were located in the difference map computed at this stage. The molecular and water H atoms were identified after further refinement and a difference synthesis at R = The final R obtained by refinement with the weighting scheme [w = 1/a(F) 2] was Scattering factors for the non-h atoms were computed with the Cromer & Waber (1965) constants, that for H was from Stewart, Davidson & Simpson (1965). Table 1. Final positional and thermal parameters for the non-h atoms ( 104) with e.s.d.'s in parentheses for x,y,z Ueq = 1/(6r?) Zt Yjflijai aj. x y z Ueq (A 2) Molecule A C(1) 4188 (8) 2867 (4) 5659 (16) 40 C(2) 4511 (6) 3384 (3) 5329 (11) 24 C(3) 5974 (7) 3622 (3) 6623 (11) 25 O(31) 6228 (5) 4102 (2) 6215 (9) 39 0(32) 6929 (6) 3266 (2) 8211 (9) 35 O(1) 3521 (5) 3801 (2) 3829 (8) 29 P(I) 2400 (2) 3744 (1) 970 (3) 21 0(2) 1672 (5) 4308 (2) 313 (9) 28 0(3) 3138 (5) 3579 (2) -238 (9) 35 0(4) 1387 (5) 3244 (2) 576 (9) 33 Molecule B C(1) (8) 2912 (3) 4829 (16) 39 C(2) (6) 3437 (3) 3924 (12) 24 C(3) (7) 3621 (3) 2207 (12) 24 O(31) (5) 4103 (2) 1326 (9) 33 0(32) (5) 3228 (2) 1808 (9) 33 O(I) -956 (5) 3884 (2) 4554 (8) 26 P(I) 129 (2) 3915 (1) 3726 (3) 21 0(2) 751 (5) 4503 (2) 4478 (9) 32 0(3) 0(4) -637 (5) 1216 (5) 3764 (2) 3419 (2) 1088 (8) 5097 (9) Na(l) 2372 (3) 4961 (1) 3711 (6) 36 Na(2) 4357 (3) 416 (1) 3500 (6) 40 W(1) 4261 (6) 5082 (2) 3128 (10) 39 W(2) 7877 (6) 4998 (2) 9677 (10) 36 Results and discussion The final positional parameters for the non-h atoms are given in Table 1, those for H in Table 2.* Bond lengths and angles are listed in Tables 3 and 4 respectively. The torsion angles are given in Table 5. * Lists of structure factors and anisotropic thermal parameters have been deposited with the British Library Lending Division as Supplementary Publication No. SUP (15 pp.). Copies may be obtained through The Executive Secretary, International Union of Crystallography, 5 Abbey Square, Chester CH 1 2HU, England. Table 2. Final positional and thermal parameters for the H atoms (x 103) x y z U (A 2) Molecule A H(1) H'(1) H(32) H(4) Molecule B n(1) H'(1) H(32) H(4) HW(1) H'W(I) HW(2) H' W(2) c(1)-c(2) c(2)-c(3) c(3)-o(31) c(3)-o(32) c(2)-o(1) O(1)-P(1) P(1)-O(2) P(1)--O(3) P(1)--O(4) Table 3. Bond lengths (A) C(1)-C(2)-C(3) C(I)-C(2)-0(I) 0(I)-C(2)-C(3) C(2)-C(3)-O(31) C(2)-C(3)-O(32) O(31)-C(3)-O(32) C(2)-O(1)-P(1) O(1)-P(1)--O(2) 0(I)--P(I)--0(3) O(1)-P(1)--O(4) O(2)-P(1)--O(3) O(2)-P(1)-O(4) O(3)-P(1)-O(4) A (11) (I 1) (9) (9) (8) (5) (5) (7) (6) Table 4. Bond angles (o) (7) (7) (6) (7) (6) (7) (5) (3) (3) (3) 117.4(3) 111.6(3) (3) B (10) (1 l) (8) (10) (9) (6) (6) (5) (6) (7) (7) (6) (7) (6) (7) (5) (3) (3) (3) (3) (3) (3)

3 836 THE STRUCTURE OF MONOSODIUM PHOSPHOENOLPYRUVATE Table 5. Torsion angles (o) involving non-h atoms Average e.s.d, is 0.3. A B C(1)-C(2)-C(3)-O(31) O(1)-C(2)-C(3)-O(31) C( 1)-C (2)-C(3)-O(32) O(1)-C(2)-C(3)-O(32) C(1)-C(2)-O(1)-P(1) C(3)-C(2)-O(1)-P(1) C(2)-O(1)-P(1)-O(2) C(2)--O(1)-P(1)-O(4) C(2)-O(1)-P(1)-O(3) angle being When viewed down P(1)-O(1), the phosphates in A and B are found to assume the normally preferred staggered conformation (Fig. 1). The phosphate geometry of both molecules deviates significantly from that of a regular tetrahedral arrangement. The O-P-O angles are distributed on either side of 109.5, the angle contained by the shortest bonds being the largest. The two independent phosphates A and B have similar bond lengths and angles, the maximum differences being for the angles 0(2)- P(1)--O(4) [111.6 (A), (B)] and O(3)-P(1)- O(4)[107.8 (A), (B), e.s.d. = 0.3 ]. Molecular geometry There are two molecules of PEP in the asymmetric unit which will be referred to as A and B. The phosphate group As the compound is a monosodium salt, the phosphate groups of both A and B are expected to carry a single negative charge. The longest of the three bond distances from P to the terminal O atoms [P(1)-O(4) = (A), 1.564A (B)] is close to the mean P-O(H) distance of 1.568/k in putrescine diphosphate (Takusagawa & Koetzle, 1978). The other two [P(1)-O(2) = 1.483, P(1)-O(3)= (A)and 1.493, 1.495/k (B)] are almost equal. Hence it is likely that the single anionic charge is distributed between 0(2) and O(3), and that the H atom is attached to 0(4). The high energy P ~ O bond is in A and ]k in B, which is comparable to that found in the monopotassium salt of PEP [ (6) A]. The H atom on 0(4) of the phosphates in A and B was located in the difference map. It was found to take up a staggered position which is more prominent in A than in B [O(1)-P(1)-O(4)-H(4) = 49.4 (A),-21.9 (B)]. The hydroxyl 0(4) is gauche with respect to C(2) in both molecules [O(4)-P(1)-O(1)-C(2) = 64.1 (A), (B)], as also found in PEP.K (-46.5 ). In contrast, it is trans in PEP-cyclohexylammonium salt (PEP.CHA) (Watson & Kennard, 1973), the torsion ~ ) 0(2) 0(3)~ "" 0 0(3) (a) (b) Fig. 1. View down the P(1)-O(1) bond of (a) molecule A, (b) molecule B. The enolpyruvate group The enolic bond. The enolic bond C(2)-O(1) is in A and 1.386/~ in B. It is comparable to the average C(sp2)-O distance of A (Newton & Campbell, 1974). Hence the enolic bond has a considerable amount of double-bond character. In PEP.K, this bond length is A. The P-O-C angle is in A and in B (e.s.d. = 0.5 ), considerably larger than the 120 normally observed in phosphate ester structures. The enolpyruvate geometry Results of mean-plane calculations for the enolpyruvate parts of the two PEP. Na molecules are given Table 6. Least-squares planes and deviations of atoms (A) Average e.s.d, in the atom deviation is 0.007,a,. A B (I) C(1) C(2) C(3) O(31) (32) O(1) (II) C(1) C(2) C(3) O(1) (Ill) C(2) C(3) (31) (32) Equations of planes (IA) x y z = (IB) x y z = (IIA) x y z = (IIB) x y z = (IliA) x y z = (IIIB) x y z =

4 S. K. KATTI, M. V. HOSUR AND M. A. VISWAMITRA 837 in Table 6. The angle between the normals to the planes formed by C(1), C(2), C(3), O(1) and C(2), C(3), O(31), 0(32) is 4.4 in A and 7.3 in B, which compares with the 3.5 in pyruvic acid (Harata, Sakabe & Tanaka, 1977). This angle is 18.1 in Na pyruvate (Tavale, Pant & Biswas, 1961), the maximum observed in pyruvate structures studied so far. This angle is an indication of the flexibility for rotation about the central C(2)-C(3) bond, i.e. the degree of conjugation across it. In PEP.Na, the C(2)-C(3) bond is A in both A and B (e.s.d. = A), suggesting partial double-bond character. It is significantly different from the 1.579,/k in Na pyruvate, which is close to the C-C single-bond length. The C=O and C-O(H) lengths (1.22 and 1.31 A) in the carboxyl group compare well with those usually found in carboxylic acid structures (Leiserowitz, 1976). The carbonyl O atom is trans planar to the olefinic C atom [C(1)-C(2)- C(3)-O(31) = (A), (B)] in both molecules. This is in contrast to the eis planar orientation in PEP.CHA (7.2 ). The angle C(3)-C(2)-O(1) is in A and in B, considerably smaller than the 120 expected at an sp 2 hybridized C atom. five ligands. It binds to three independent PEP's and two waters W(2) and W(2)'. Somewhat similar six and five coordinations for Na ions have been observed in 5'-dGMP.Na2 (Young, Tollin & Wilson, 1974; Viswamitra & Seshadri, 1974). The ester O(1) of A is 2.98 A from Na(1) and that of B is 2.92 ]k from Na(2). In PEP. K, the K + ion is almost in the plane of the enolpyruvate (deviation = 0.02 A) and binds to the phosphate, ester and carboxyl O atoms of the same molecule. However, in the present structure, Na is involved in bridging only the phosphate ends of different molecules. The Na ion, because it is smaller (ionic radius = 0.95 A; Pauling, 1960), is not able to link both the carboxyl and phosphate ends of the same molecule. It is probable that the differences in the molecular shapes of PEP. Na and PEP. K are brought about by the differences in their metal-ion binding patterns. Molecular packing and hydrogen bonding There are four intermolecular hydrogen bonds in the structure. Two of them link the PEP molecules along a Orientation of the phosphate relative to the enolpyruvate system The orientation of the phosphate with respect to the enolpyruvate system in A is different from that in B, the torsion angle C(3)-C(2)-O(1)-P(1) being in A and in B. This makes the molecular shapes of A and B almost mirror images of each other, the mirror being the planar enolpyruvate group (Fig. 2). This dihedral angle is in PEP.K and -90 in PEP. CHA. ~...~)o(3,) o(3(~ c(1)~ ~ ~ c(~) o(3)t ~.~j ~ (~;)o(i) I x\ O(32K~ T ".,.,/~ ~r~o Na + ion coordination There are two crystallographicauy independent Na + Fig. 3. Packing diagram down e. ions in the structure. The nature of their interactions with the surroundings is different. Na(1)has six nearest _,,,,--1. neighbours. Four of them are contributed by the f ~ -.--, ~ 1~ phosphates of four different PEP molecules and the remaining two by the water molecule W(1) and its,~ symmetry equivalent W(1)'. Na(2) is coordinated to q x. o,,o C ( 1)~'~'~'#,I ~.~(~4) 1". k (a) (b),, Fig. 2. View along the enolpyruvate plane of (a) molecule A, (b) ' molecule B. Fig. 4. Stere view of the molecular packing.

5 838 THE STRUCTURE OF MONOSODIUM PHOSPHOENOLPYRUVATE O(3B)... H(32)--O(32) (3~O(31)... W(2) O(3A) - 71,... wi)) H(32)-O(32) ~O(31) CT(3) '~f "~"~ W(2) Ci2) 2)~.Hit) / \ /Hil) Oit) [ c()lh'(') O(,) [ c(,) "H'(,) Na(2)*.~...~,P(I).~.-Na( ) Oi4B)... O(3)/~%1 O(2>:......j.- Na, )Nail). l O'4A)... ~O(3) l ""Oi2) ~. Nai2 )..-"" O(41 "'-.. ~.-" 0(4) "'~._. " I "~" Na(2) "" I ~r~a( 1)* O(32BY H(4) 0(32,4) "/ H(4) i i 6(3B)* O(3A)* t (a) (b) Fig. 5. Schematic diagram showing the molecular interactions of (a) molecule A, (b) molecule B. An asterisk marks atoms related by a different symmetry element of the space group. Table 7. Sodium coordination and hydrogen bonding (A) E structure is completed through metal-ion binding and the hydrogen bonds involving the water molecules (Table 7). Fig. 4 shows the molecular packing. Fig. 5 is a schematic diagram illustrating the molecular interactions of the two individual molecules A and B. Carboxylic acids generally form dimeric structures through hydrogen bonds between the carbonyl and hydroxyl O atoms (Leiserowitz, 1976). Pyruvic acid shows this type of association in its crystal structure. In the present structure, however, the carboxyl groups are not involved in any dimer formation. Instead, they stack one above the other at an average distance of 3.2A. We thank the DST for financial support. One of us (SKK) thanks the CSIR for the award of a Fellowship. Average e.s.d, is A. Na(1).. O(2A) Na(1).. O(2B) Na(1).. O(2B) Na(1).. O(2A) Na(1).. W(1) Na(1).. W(1) Na(2)...O(2B) Na(2)...O(3B) Na(2)...O(2A) Na(2)... W(2) Na(2)... W(2) O(3A)...O(32B) O(4B)...O(3A) O(32A)...O(3B) O(4A)...O(3B) 0(31A).. W(2) 0(31B)... W(2) O(31B)... W(1) Symmetry code I I Symmetry code: (1) x,y,z; (2) ½ + x, ½ + y, z; (3) x, ), ½ + z; (4) ½ + x, ½ - y, ½ + z. The numbers following the symmetry code indicate cell translations of the atom. [O(3A)...H-O(32B) = 2.57, O(4A)--H...O(3B)= 2.79 ~] and the other two along e [O(4B)-H... O(3A) = 2.64, O(32A)-H...O(3B) = 2.61 A) as shown in the packing diagram (Fig. 3). The extended crystal References CROMER, D. T. & WABER, J. T. (1965). Acta C~st. 18, HARATA, K., SAKABE, N. ~ TANAKA, J. (1977). acla Crysl. B33, HOSUR, M. V. & VISWAMITRA, M. A. (1981). Acta Cryst. B37, LEISEROWlTZ, L. (1976). Acta Cryst. B32, MAHLER, H. R. & CORDES, E. H. (1969). In Basic Biological Chemistry. New York: Harper & Row. NEWTON, M. G. & CAMPBELL, B. S. (1974). J. Am. Chem. Soc. 96, PAULING, L. (1960). The Nature of the Chemical Bond, p Ithaca: Cornell Univ. Press. SHELDRICK, G. M. (1976). SHELX 76. Program for crystal structure determination. Univ. of Cambridge, England. STEWART, R. F., DAVIDSON, E. R. ~,~ SIMPSON, W. T. (1965). J. Chem. Phys. 42, TAKUSAGAWA, F. & KOETZLE, T. F. (1978). Acta Cryst. B34, TAVALE, S. S., PANT, L. M. & BISWAS, A. B. (1961). Acta Cryst. 14, VISWAMITRA, M. A. & SESHADRI, T. P. (1974). Nature (London), 252, WATSON, D. G. & KENNARD, O. (1973). Acta Cryst. B29, YOUNG, O. W., TOLLIN, P. & WILSON, H. R. (1974). Nature (London), 248,